Herein is proposed an strategy for expanding the genetic alphabet via engineering unnatural base pair acceptance by DNA polymerases. This research proposal addresses the following specific aims: 1) to engineer a DNA polymerase that processes unnatural base pairs with high fidelity, 2) to identify the structure-function relationships that dictate polymerase fidelity and processivity, and 3) to clarify the role of hydrogen bonding with respect to base pair stability. The incorporation of any of the proposed base pairs will create 152 additional codons that can be applied to broader studies of protein structure and function within the Schultz group. This effort will provide unprecedented access to the incorporation of multiple unnatural amino acids into a single protein sequence, the implications of which are numerous. The successful incorporation of unnatural base pairs by engineered DNA polymerases will represent a landmark achievement and will significantly advance the frontiers of science and medicine.